TY - JOUR
T1 - Profiling the phospho-status of the BKca channel α subunit in rat brain reveals unexpected patterns and complexity
AU - Yan, Jiusheng
AU - Olsen, Jesper V.
AU - Park, Kang Sik
AU - Li, Weiyan
AU - Bildl, Wolfgang
AU - Schulte, Uwe
AU - Aldrich, Richard W.
AU - Fakler, Bernd
AU - Trimmer, James
PY - 2008/11
Y1 - 2008/11
N2 - Molecular diversity of Ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BKca) channels are tetramers of α subunits (BKα) either alone or together with β subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca2+. The cytoplasmic C terminus of BKα is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanof low liquid chromatography tandem mass spectrometry on BKca channels affinity-purified from rat brain to analyze in vivo BKα phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho-and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKα differentially modulates the voltage- and Ca2+ -dependence of channel activation. These results demonstrate that the pore-forming subunit of BKca channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKα structure and function.
AB - Molecular diversity of Ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BKca) channels are tetramers of α subunits (BKα) either alone or together with β subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca2+. The cytoplasmic C terminus of BKα is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanof low liquid chromatography tandem mass spectrometry on BKca channels affinity-purified from rat brain to analyze in vivo BKα phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho-and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKα differentially modulates the voltage- and Ca2+ -dependence of channel activation. These results demonstrate that the pore-forming subunit of BKca channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKα structure and function.
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U2 - 10.1074/mcp.M800063-MCP200
DO - 10.1074/mcp.M800063-MCP200
M3 - Article
C2 - 18573811
AN - SCOPUS:52049111074
VL - 7
SP - 2188
EP - 2198
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 11
ER -